The long term aim of this project is to understand the mechanism by which specific interactions within proteins contribute to their overall stability. To this end, the goal of the proposed research is to identify and quantitate native-state interactions that are responsible for stability of ovomucoid third domain, a small globular protein. Using hydrogen exchange, the aim understand how highly localized changes in sequence, such as a single amino acid substitutions, can lead to global changes in protein stability. Stabilizing interactions will be identified by correlation of hydrogen exchange behavior with the thermodynamics of third domain folding. The first aim is examination of the behavior of amide protons (NHs) are likely to be exchanging only when the protein is completely unfolded and, hence, ar reporting on the most stable regions in ovomucoid third domain. The second aim is an investigation of the role of specific electrostatic interactions in third domain stability. The third aim is quantitation of additional types of specific interactions through thermodynamic and hydrogen exchange studies of third domain variants. Identification and quantitation of specific stabilizing interactions in proteins will increase our understanding of the factors responsible for stability. This will, in turn, lead to a) improved design of proteins with increased stability, b) enhanced capabilities for the design of new proteins, c) a valuable quantitative data base for theoreticians, and d) a better understanding of how the amino acid sequence dictates the folding and stability of proteins.